KR20110093474A - Composition for composition for prevention or treatment of rotavirus infection comprising glycyrrhiza uralensis - Google Patents

Abstract

PURPOSE: A composition containing Glycyrrhiza uralensis Fischer extract or fraction is provided to ensure anti-rotavirus effect and to prevent or treat rotavirus infection. CONSTITUTION: A composition for preventing or treating rotavirus infection contains Glycyrrhiza uralensis Fischer extract or fraction as an active ingredient. The extract is isolated using water, alcohol of C1-C4, or mixture solvent thereof. The fraction is a hexane, ethyl acetate, or water fraction. The rotavirus is human porcine, bovine, or goat rotavirus. The composition is a pharmaceutical or food composition. The pharmaceutical composition is used in the form of tablet, powder, pill, granule, capsule, suspension, emulsion, syrup, suspension, or suppository.

Description

Composition for composition for prevention or treatment of rotavirus infection comprising glycyrrhiza uralensis}

The present invention relates to a composition for the prevention and treatment of rotavirus infection comprising licorice extract, fractions, or polyphenolic compounds isolated therefrom.

Rotavirus is one of the most important causes of severe diarrhea in young animals (cows, pigs, and goats) and infants around the world.In addition to severe diarrhea, rotaviruses include encephalitis, otitis media, necrotic colitis, liver abscess and intestinal tract. It is known to cause disease (Ester, MK Rotaviruses and Their Replication in Fields Virology. Pp. 1747-1785, 2001). Rotavirus belongs to the reoviridae family and surrounds a 11-segmented double-stranded RNA genome with a diameter of 70 mm surrounded by three capsid layers. The outermost of these three capsid layers is composed of VP4 and VP7 proteins. Since the VP4 protein is divided into VP5 and VP8 by the protease of the host, its serotype is called P type. Since the VP7 protein is glycosylated, its serotype is called G type (Ciarlet, M. and Ester, MK Rotaviruses: basic biology, epidemiology and methodologies in encyclopedia of envirionmental microbiology.pp. 2753-2773, 2002). VP4 and VP7 proteins are critically involved in the defense of the host by inducing neutralizing antibodies.

The serotypes were divided into a total of seven species up to A-G by serotypes for VP6, the interlayer capsid protein. Of these, serotype A rotavirus is the most common in humans and animals, causing 130 million people worldwide each year, of which 873,000 are known to be the most important infectious agents of death.

Serotype A rotavirus has been reported with G and P types specific to each host. In humans, the combination of G1-G3, G9 and P [4] and P [8] is most prevalent (Kapikian et al., 2001). In pigs, the combination of G3-G5, G11 and P [6] and P [7] is known to be most prevalent (Gouvea and Timenetsky, 1994). Cattle are favored by a combination of G6, G8, G10 and P [1], P [5], and P [11] (Alfieri et al., 2004). However, similar to the influenza virus, there has been a reported case of a recombinant virus occurring in humans as a result of a mixed infection of human and animal serotype A rotavirus in humans with frequent physical contact with animals. (jain et al., 2001). In addition, in Brazil and the United States, G9 rotavirus, which occurs mainly in humans, also occurs in pigs (Paul et al., 1988). The detection of rotaviruses derived from animals in humans means that they are "causal agents of common communicable disease," and that breeding between breeding stocks, which can be transmitted to other livestock, is also occurring.

In general, for the purpose of treating such viral diseases, methods such as inhibition of adsorption into epithelial cells, inhibition of invasion into cells, inhibition of gene transcription and replication, inhibition of protein synthesis, and inhibition of release from cells can be considered. Although reported as an antiviral target, practically no clear targets for rotavirus have been identified.

Many vaccines have been developed to suppress the infection of rotavirus, and in particular, humans produce and sell animal-human recombinant virus vaccines made by recombination of human and animal rotaviruses. Representatively, a vaccine containing VP4 gene, which is based on bovine rotavirus strain WC3, having the specificity of human rotavirus strain serotype, and RotaTeq (Merk Co., Ltd.) received US FDA approval in 2006. It is sold. However, such a recombinant virus vaccine has not only reported side effects such as intussusception, but also has a big disadvantage that it cannot prevent against various rotavirus serotypes.

In order to overcome the disadvantages of these vaccines, research on natural materials that can inhibit rotavirus has been made. Takahashi et al. Reported that stevia hydrothermal extracts have anti-rotavirus effects (Takahashi, K et al., Antiviral Res. 49, 15, 2001).

Therefore, the research team confirmed that licorice extract, fractions, or compounds isolated therefrom have the activity of inhibiting rotavirus infection, and have the effect of inhibiting killer virus and cytopathic effect on rotavirus. Reached.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of rotavirus infection and a method of treatment using the same.

Another object of the present invention is to provide a food composition for the prevention or treatment of rotavirus infection.

Another object of the present invention is to provide a quasi-drug composition for the prevention or treatment of rotavirus infection.

In one aspect of the present invention for achieving the above object, there is provided a composition for the prevention or treatment of rotavirus infection comprising licorice extract or fractions thereof as an active ingredient.

According to another aspect of the invention, there is provided a composition for the prevention or treatment of rotavirus infection comprising any one of the compounds represented by the formula 1 to 5 as an active ingredient.

[Formula 1]

[Formula 2]

(3)

[Formula 4]

[Chemical Formula 5]

According to another aspect of the invention, there is provided a method of inhibiting the activity of rotavirus comprising contacting said composition with rotavirus.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for preventing or treating rotavirus infection comprising licorice extract or fractions thereof as an active ingredient.

The composition of the present invention can be used for the prevention or treatment of rotavirus infection because it exhibits a virucidal and cytopathic inhibitory effect on rotavirus at the same time.

As used herein, the term "prevention" means any action that inhibits or delays the onset of rotavirus infection by administration of a composition.

As used herein, the term "treatment" means any action that improves or advantageously changes the symptoms caused by rotavirus infection by administration of the composition.

The rotavirus may be human, pig, bovine, or goat rotavirus. The rotavirus may also cause diseases of enteritis, diarrhea, colds, sore throat, bronchitis, and pneumonia.

Licorice in general ( Glycyrrhiza uralensis is a perennial herb that belongs to the legume and grows or grows in northern China, Siberia, southern Italy, Manchuria, and Mongolia. Licorice is one of the herbs used as an anti-tussive agent in all herbal remedies and used as an antitussive (Rauchensteiner F. et al., JPharm Biomed Anal, 38 (4), pp.594-600, 2005). It can also be used to sweeten candy. Recent studies have shown that licorice mitigates oxidative damage in the kidneys (Yokozawa T. etal., FreeRadic Res., 39 (2), pp.203-211, 2005). (Kim SC. Et al., Toxicology., 197 (3), pp.239-251, 2004).

Licorice in the present invention can be used to buy a commercially available, or directly harvested or grown in nature.

The method of preparing the extract of licorice may use conventional extraction methods in the art, such as ultrasonic extraction, filtration and reflux extraction. Preferably, the licorice dried material obtained by pulverizing the licorice from which foreign substances are removed by washing and drying may be an extract extracted with water, alcohol of C ₁ to C ₄ or a mixed solvent thereof, and more preferably, C ₁ to C ₄ . It may be an extract extracted with alcohol, most preferably may be an extract extracted with methanol or ethanol. At this time, the extraction solvent is preferably 2 to 20 times the dry weight of licorice. For example, the licorice dry matter is chopped, and then put into an extraction container, a lower alcohol of C ₁ to C ₄ or a mixed solvent thereof, preferably methanol or ethanol, and allowed to stand at room temperature for a certain period of time, followed by filtration to obtain an alcohol extract. . At this time, the extraction is preferably left for 1 week at room temperature, after which it may be further subjected to methods such as concentration or lyophilization.

On the other hand, licorice fraction in the present invention can be obtained by fractionation from the licorice extract obtained above. More specifically, the licorice fraction may be obtained by suspending the licorice extract in water and then fractionating with hexane and ethyl acetate, respectively, to obtain a hexane fraction, an ethyl acetate fraction and a water fraction.

Furthermore, the present invention provides a composition for preventing or treating rotavirus comprising any one of the compounds represented by the following Chemical Formulas 1 to 5 as an active ingredient.

[Formula 1]

[Formula 2]

(3)

[Formula 4]

[Chemical Formula 5]

The compounds represented by Formulas 1 to 5 according to the present invention are representative active ingredients present in licorice, and the method for separating the compounds may be performed as follows.

First, licorice is extracted with water, C ₁ -C ₄ alcohol or a mixed solvent thereof to obtain a licorice extract (step 1). The licorice can be used without limitation, such as cultivated or commercially available, it is used to clean and dry. The alcohol may be a lower alcohol such as methanol, ethanol, propanol, butanol, preferably methanol or ethanol.

In the step 1, the step of obtaining the licorice extract is to make the dried licorice powder in order to increase the efficiency of extracting the shade of the shade, put it in an extraction container and add an appropriate amount of alcohol, and leave it at room temperature for 5 days Filter paper may be carried out by filtration. This extraction process may be repeated several times, and then a method such as concentration or lyophilization may be additionally performed.

Subsequently, water is added to the licorice extract obtained in step 1, suspended, and fractionated sequentially using hexane and ethyl acetate to obtain a licorice fraction (step 2). At this time, a conventional fractional extraction method can be used, and preferably a fractional funnel can be used. The licorice fraction can be obtained as a hexane fraction, ethyl acetate fraction, butanol fraction, and water fraction.

Thereafter, the licorice fraction obtained in step 2 may be purified by separating and purifying the compound by silica gel chromatography (step 3). When performing silica gel chromatography for separating the compounds, it is preferable to use n -hexane, n-hexane ethyl acetate, a chloroform-acetone mixed solvent and methanol as the mobile phase, and n -hexane / acetone mixed in the additional chromatography. Solvents may be used. The volume ratio of the n -hexane / ethyl acetate mixed solvent used at this time is 50: 1-1: 1 volume ratio, and 150: 1-1: 1 volume ratio is preferable when it is a chloroform and acetone mixed solvent. The chromatography may be performed once or several times until a single compound is purified, and may be concentrated and recrystallized as necessary.

In addition, the compounds represented by Chemical Formulas 1 to 5 of the present invention may be used in the form of pharmaceutically acceptable salts, respectively, and as salts, acid addition salts formed by pharmaceutically acceptable free acid are useful. Do. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salts according to the present invention can be dissolved in conventional methods, for example, by dissolving the above formula (1) in an excess of aqueous acid solution and using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation.

In addition, the compounds represented by Chemical Formulas 1 to 5 of the present invention may use a base to form a pharmaceutically acceptable metal salt. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding silver salts are also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg silver nitrate).

Meanwhile, the composition for preventing or treating rotavirus infection of the present invention may be a pharmaceutical composition.

When the composition of the present invention is a pharmaceutical composition, the composition may include a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose ( lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. It can have one formulation.

The composition of the present invention is administered in a pharmaceutically effective amount.

As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level refers to the individual type and severity, age, sex, type of virus infected. , The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the concurrent drug and other factors well known in the medical field. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

The composition of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, drug treatment and biological response modifiers for the prevention or treatment of rotavirus infection.

On the other hand, the present invention provides a method for treating a rotavirus infection disease comprising administering the composition for the prevention or treatment of rotavirus infection in a pharmaceutically effective amount to a subject having the invention or the possibility of rotavirus infection. . The rotavirus may be human, pig, bovine, or goat rotavirus. In addition, the rotavirus infection disease may be enteritis, diarrhea, cold, sore throat, bronchitis, and pneumonia.

As used herein, the term "individual" means all animals, including humans already infected with or capable of being infected with a rotavirus, and by effectively administering to a subject a composition comprising an extract or fraction of the invention, thereby effectively preventing and treating the disease. Can be. For example, the compositions of the present invention can treat humans infected with various rotavirus subtypes or variants of human rotavirus. In addition, the compositions of the present invention can treat humans infected with various rotavirus subtypes or variants of rotavirus. It is also possible to treat pigs, cattle, or goats infected with various rotavirus subtypes or variants of rotavirus. The composition of the present invention can be administered in parallel with a therapeutic agent for existing rotavirus infection disease.

The route of administration of the composition may be administered via any general route as long as it can reach the desired tissue. The composition of the present invention may be administered as desired, but is not limited to intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, pulmonary administration, rectal administration. The composition may also be administered by any device in which the active agent may migrate to the target cell.

In addition, the present invention provides a food composition for the prevention or treatment of rotavirus infection comprising any one of the licorice extract, fractions, or compounds represented by the following formula 1 to 5 isolated therefrom as an active ingredient.

[Formula 1]

[Formula 2]

(3)

[Formula 4]

[Chemical Formula 5]

That is, the licorice extract, fractions of the present invention, or the compounds represented by Formula 1 to 5 separated therefrom may be added to the food composition for the prevention or treatment of rotavirus infection. When the licorice extract of the present invention or a fraction thereof is used as a food additive, the extract or fraction may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient can be determined suitably according to the purpose of use (prevention, health or therapeutic treatment). In general, in the manufacture of food or beverages, the composition of the present invention is added in an amount of 0.01 to 10% by weight, preferably 0.05 to 1% by weight based on the raw materials. However, in the case of prolonged ingestion for health and hygiene purposes or for health control purposes, the amount may be used below the above range.

There is no particular limitation on the kind of food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And a carbonation agent used for the carbonated beverage. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention. These components can be used independently or in combination.

In addition, the present invention provides a quasi-drug composition for the prevention or treatment of rotavirus infection comprising any one of licorice extract, fraction, or a compound represented by the following formula (1) to 5 as an active ingredient.

[Formula 1]

[Formula 2]

(3)

[Formula 4]

[Chemical Formula 5]

That is, the composition of the present invention may be added to the quasi-drug composition for the purpose of preventing or treating rotavirus infection. When the licorice extract, fraction, or compound isolated therefrom of the present invention is used as an quasi-drug additive, the extract or fraction may be added as it is or used with other quasi-drug components, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).

Preferably, the quasi-drug composition may be an antiseptic agent, a shower foam, a gagreen, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment, or a filter filler.

The present invention also provides a method of inhibiting the activity of rotavirus comprising contacting the composition with rotavirus. At this time, the contact is made in a conventional manner, rotavirus includes a living organism, tissue or cell culture, biological material samples (blood, serum, urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples, etc.), Usually it may contain pathogenic organisms such as viruses. Such samples can be contained in any medium containing water and an organic solvent / water mixture.

It can also be observed by any method, including direct or indirect methods of diagnosing anti-rotavirus activity after administration of the compositions of the present invention. Either quantitative, qualitative or semi-quantitative methods of diagnosing rotavirus infection inhibitory activity are available, and any other method may be applied, such as observing the physiological properties of living organisms.

Licorice extract, fractions, or compositions comprising a polyphenolic compound isolated therefrom according to the present invention exhibits the effect of inhibiting the activity of rotavirus, and simultaneously shows the effect of inhibiting virucidal and cytopathic activity against various rotaviruses. It can be usefully used for the prevention or treatment of rotavirus infection.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the following examples are merely to illustrate the present invention is not limited to the contents of the present invention.

Example  One : licorice  Preparation of Extract

10 L of 100% ethanol (EtOH) was added to 2 kg of licorice root purchased from Jeongeup, Jeollabuk-do, Korea, and left for 3 days at room temperature. The resultant was filtered through a filter paper and concentrated to obtain an ethanol extract of licorice (134 g).

Example  2: from licorice extract Fraction  And Polyphenolic  Isolation and Purification of the Compound

1 L of water was added to 134 g of brown powder obtained from the ethanol extract of the licorice root obtained in Example 1, and suspended. Put it in a separatory funnel, and fractionally extract using n -hexane, chloroform and ethyl acetate in this order. N -hexane soluble extract (1.3 g), chloroform soluble extract (45 g), ethyl acetate soluble extract (35 g) and water Soluble extract was obtained.

Silica gel column chromatography (silica gel 500 g, 70-230 mesh) using 20 g of the chloroform soluble extract obtained above as a mobile phase with 100% chloroform, acetone and a mixed solvent thereof (40: 1 to 1: 1) ) Was separated into 10 fractions (Fr. 1-10). The third of these fractions (Fr. 3, 3 g) was prepared again by silica gel column chromatography (7 g, using a mixed solvent of n -hexane: ethyl acetate (40: 1 to 2: 1 (v / v)) as a mobile phase. 230-400 mesh) was carried out, thereby obtaining 30 fractions (Fr. 3-1-30). Among them, preparative TLC (Preparative TLC) was performed on Fr.3-18-20 fractions (0.11 g), using compound 5 using a solvent of n -hexane: ethyl acetate = 4: 1 (v / v) as a mobile phase. (13 mg) was obtained.

In addition, silica gel column chromatography (silica gel 500 g, 70 to 230 mesh (mesh: 17 g) of the ethyl acetate soluble extract was prepared using 100% n -hexane, ethyl acetate and a mixed solvent thereof (60: 1 to 1: 4) as a mobile phase. )) To separate 14 fractions (Fr. 1-14). The second of these fractions (Fr. 2, 1.5 g) was purified by silica gel column chromatography (30 g, 30%, n -hexane: ethyl acetate (50: 1 to 2: 1 (v / v)) as a mobile solvent. 230 to 400 mesh) was performed, thereby obtaining 10 fractions (Fr. 3-1 to 10). Among them, Fr.3-4 fraction (0.21 g) was subjected to column using Sephadex-LH20 to obtain Compound 4 (32 mg), and the same column chromatography was also performed on Fr.3-8 fraction. Compound 1 (13 mg) was obtained.

In addition, silica gel column chromatography (40 g, 230 ~) using n -hexane: ethyl acetate (30: 1 ~ 1: 2) as the mobile phase solvent for the tenth fraction (Fr.10, 1.2 g) of the chloroform soluble extract. 400 mesh) was carried out to obtain 20 fractions (Fr. 10-1-20). Among them, silica gel column chromatography was performed again on the fractions of Fr. 10-12 to 15 to obtain Compound 1 (14 mg) and Compound 2 (54 mg).

In addition, Sephadex-LH20 and silica gel column chromatography were sequentially performed on the fractions Fr. 10-15 to 20 of the chloroform soluble extract to obtain compound 3 (20 mg).

Example  3: structural analysis of compounds

The molecular weight and molecular formula of the compound obtained in Example 2 were determined using a VG high resolution GC / MS spectrometer, Election Ionization MS, Autospec-Ultima. In addition, molecular structure was determined using spectroscopic data of ¹ H NMR and ¹³ C NMR through nuclear magnetic resonance (NMR) analysis (Bruker AM 300, 500).

As a result of comparing the results of the above instrumental analysis with that of the published literature, isoliquiritigenin represented by the following formula (1), 2-methoxyisoliquitin represented by the following formula (2), It was confirmed that the lycopumarone represented by the formula (3), the gliasperrin C represented by the following formula (4), and the lycoflavonol represented by the formula (5) ( J. Agric . Food Chem . 7408-7414, 2005; Phytochemistry , 287-293, 1998; Chem . Nat . Comp ., 389, 1972; Chem . Pharm . Bull . , 1286-1292, 2000). The specific analysis results were as follows.

Compound 1: Issoriquitigenin

[Formula 1]

1) Physical property: Yellow solid (m.p. 206-210 ℃)

2) Molecular Weight: 256

3) Molecular formula: C ₁₅ H ₁₂ O ₄

4) ¹ H-NMR (methanol-d ₃ , 500 MHz) δ 6.28, 6.41, 6.83, 7.59, 7.61, 7.78, 7.95. ¹³ C-NMR (methanol-d ₃ , 125 MHz) δ 193.7, 167.7, 166.6, 161.7, 145.8, 133.5, 131.9, 128.0, 118.5, 117.0, 114.8, 109.3, 103.9.

Compound 2: 2- Methoxyisosoriquitygenin

[Formula 2]

1) Physical property: Yellow solid (m.p. 89-92 ℃)

2) Molecular Weight: 270

3) Molecular formula: C ₁₆ H ₁₄ O ₄

4) ¹ H-NMR (methanol-d ₃ , 500 MHz) δ 3.78, 6.37, 6.78, 7.49, 7.86, 7.87. ¹³ C-NMR (methanol-d ₃ , 125 MHz) δ 56.5, 100.4, 109.7, 116.76, 117.2, 119.9, 131.9, 132.1, 132.5, 141.7, 162.6, 163.6, 164.0, 192.1.

Compound 3: Ricoku Marron

(3)

1) Physical property: Yellow solid (m.p. 183-185 ℃)

2) Molecular Weight: 340

3) Molecular formula: C ₂₀ H ₂₀ O ₅

4) ¹ H-NMR (acetone-d ₆ , 300 MHz) δ 1.65, 1.79, 3.42, 5.27, 6.50, 6.57, 6.78, 7.31, 7.69. ¹³ C-NMR (acetone-d ₆ , 75 MHz) δ 158.2, 155.2, 153.8, 153.3, 151.1, 150.6, 129.5, 127.1, 124.1, 114.7, 113.6, 110.1, 107.4, 103.1, 101.0, 92.4, 59.5, 25.0, 22.5, 17.0.

Compound 4: Gliasperin  C

[Formula 4]

1) Physical property: white powder

2) Molecular Weight: 356

3) Molecular formula: C ₂₁ H ₂₄ O ₅

4) ¹ H-NMR (methanol-d ₄ , 300 MHz) δ 1.50, 1.59, 2.60, 2.71, 3.08, 3.19, 3.51, 3.76, 4.01, 5.04, 5.93, 6.10, 6.16, 6.72. ¹³ C-NMR (methanol-d ₄ , 75 MHz) δ 18.3, 24.0, 26.3, 27.2, 33.2, 61.3, 71.4, 100.4, 103.9, 108.5, 109.0, 115.5, 120.7, 126.0, 129.2, 131.2, 155.2, 156.2, 157.6, 158.4, 158.8.

Compound 5: Ricoflavonol

[Chemical Formula 5]

1) Physical property: yellow powder

2) Molecular weight: 354 (m.p. 120-130 ℃)

3) Molecular formula: C ₂₀ H ₁₈ O ₆

4) ¹ H-NMR (acetone-d ₆ , 300 MHz) δ 1.66, 1.78, 3.31, 5.24, 6.42, 6.89, 8.07. ¹³ C-NMR (acetone-d ₆ , 75 MHz) δ 18.0, 22.3, 26.1, 93.7, 104.4, 112.3, 116.4, 123.7, 124.0, 130.7, 132.0, 137.2, 147.8, 156.3, 159.2, 160.6, 163.6, 177.4.

Experimental Example  1: licorice extract, Fraction  And the inhibitory effect of the compound isolated therefrom on rotavirus

First, to measure the viral and CPE reduction effect of bovine rotavirus NCDV (G6P [1]) and porcine rotavirus KJ205 (G5P [7]) of compounds isolated from licorice. in order, using the monkey kidney cell line TF-104 (Fetal rhesus monkey kidney ) in the following In vitro experiments were performed.

First, TF-104 cells were put in a 96 well microplate to be 4 X 10 ⁴ / well for each well, and cultured in medium alpha-MEM (penicillin 100 units, streptomycin 100 mg, 5% FBS). When TF-104 cells became monolayers, they were washed twice with alpha-MEM medium containing antibiotics only. NCDV and KJ205 were each diluted to 0.01 MOI and placed in EP tubes. Licorice extract and polyphenolic compound diluted with DMSO (dimethylsulfoxide) were added to each tube for each concentration, and reacted at 4 ° C. for 1 hour. After 1 hour, TF-104 cells washed in advance were inoculated at 3 wells per concentration (hereinafter, referred to as a sample treatment group). On the other hand, the non-infected + non-administered control group (cell group not infected with Control, NCDV or KJ205, but not treated with the compound) and the infection + non-administered control group (cell group not treated with the compound after being infected with Virus control, NCDV or KJ205) After reacting for 1 hour under the same conditions, inoculated to TF-104 cells and incubated for 1 hour at 37 ℃. After 1 hour, the plate medium was removed and washed once with PBS, and then 100 mL of alpha-MEM medium containing antibiotics and 1 mg / mL trypsin was added to each well, followed by 5 at 37 ° C. Incubated for ~ 6 days. Infected + non-administered controls were cultured for 5-6 days until complete cytopathic effect (CPE). Cell state was observed daily by inverted microscope. After 5-6 days of culture, 1 mL of neutral red solution (Sigma, UK) was added to each well to determine the viability of the cells, followed by reaction at 37 ° C. for 2 hours. After that, the medium solution was removed, and 100 mL of destaining soultion (1% glacial acetic acid, 49% DDW, 50% EtOH) was added to each well, and the absorbance was measured at 540 nm after 15 minutes. At this time, after comparing the virucidal effect (% lower) represented by the non-infected + non-administered control and the infection + non-administered control and the compound isolated from the licorice of the present invention by the following formula (1), the results are shown in the following table 1 is shown.

In the above formula, the OD value means absorbance value measured at 540 nm.

On the other hand, in order to investigate the cytopathic inhibitory effect of bovine rotavirus NCDV (G6P [1]) and porcine rotavirus KJ205 (G5P [7]) of compounds isolated from licorice, it was determined using alpha-MEM medium containing only antibiotics. TF-104 cells washed three times were inoculated with the virus and incubated at 37 ° C for 1 hour. After 1 hour, all the inoculated virus solution was removed, and licorice extract and polyphenolic compounds were treated to TF-104 cells inoculated with the virus by concentration. Thereafter, the cytopathic inhibitory effect (lower percent (%)) represented by the compound isolated from the licorice of the present invention by the same method as described above in Equation 1, the results are shown in Table 1 below.

The measurement results are shown in Table 1 below.

sample Virucidal effect NCDV (G6P [1]) KJ205 (G5P [7]) CC ₅₀ ^{1 )} (μM) EC ₅₀ ^{2 )} (μM) SI ³⁾ CC ₅₀ (μM) EC ₅₀ (μM) S.I. Ethanol extract 368.6 - - 368.6 22.4 16.4 Compound 3 - - - 59.6 26.1 2.3 Compound 4 39.1 26.2 1.5 39.1 18.7 2.1 sample CPE inhibition effect NCDV (G6P [1]) KJ205 (G5P [7]) CC ₅₀ (μM) EC ₅₀ (μM) S.I. CC ₅₀ (μM) EC ₅₀ (μM) S.I. Ethanol extract 368.6 118.7 3.1 368.6 27.4 13.5 Compound 1 - - - 137.9 35.3 3.9 Compound 2 - - - 279.9 24.0 11.7 Compound 3 - - - 59.6 26.1 2.3 1) 50% concentration value from cytotoxic concentration, 2) 50% concentration value from inhibitory effect concentration, 3) Selection index, CC ₅₀ / EC ₅₀

As shown in Table 1, it was confirmed that the licorice extract of the present invention and compounds isolated therefrom exhibited excellent inhibitory activity against various rotavirus strains. Licorice extract and Compounds 3 and 4 isolated therefrom showed a very good virucidal effect with a seletive index (SI) of 1.5 to 16.4. Compound 3 showed selective virucidal activity only in porcine rotavirus, while compound 4 showed good activity in both bovine and porcine rotavirus. Therefore, extracts and compounds isolated from licorice could be confirmed to have inhibitory activity against various rotaviruses.

In addition, as shown in Table 1, the licorice extract showed a selection index of 3.1 in bovine rotavirus and 13.5 in pig rotavirus, showing the effect of cytopathic inhibition against various rotaviruses. From this, it was confirmed that the compounds 1 to 3 exhibited an excellent cytopathic inhibitory effect against porcine rotavirus. In particular, Compound 2 was found to have a very good activity of 279.9 μM of CC ₅₀ , 24.0 μM of EC ₅₀ , and 11.7 of choice index, showing very low toxicity and high virus inhibitory effect.

Therefore, the composition of the present invention shows a virucidal effect that acts directly on the virus before the virus is infected with the cell, and an excellent inhibitory effect on cytopathic inhibition that inhibits the replication process after the virus is infected with the cell. It was confirmed that it can be usefully used for the prevention and treatment of diseases.

Experimental Example  2: Acute Toxicity Test of Compounds 1-5

In order to determine the acute toxicity of the compounds obtained in Example 2, the following experiment was performed.

SPF (specific pathogens free) 6-week-old C57BL / 6J mice were divided into 4 groups (3 males and 3 females / experimental group) of 12 male and female, temperature 22 ± 3 ℃, humidity 55 ± 10%, illumination 12 L / It was bred in a 12D animal room. Mice were allowed to acclimate for about 1 week prior to use in the experiment. Feed for experimental animals (for mice and rats, Cheil Jedang Co., Seoul, South Korea) and drinking water were sterilized and supplied freely.

Representatively, Compound 2 prepared in Example 2 was prepared in 0.5% tween 80 at a concentration of 50 mg / mL, followed by 0 mL, 0.04 mL (100 mg / kg), 0.2 mL per 20 g of mouse body weight. (500 mg / kg) and 0.4 mL (1,000 mg / kg) were orally administered to each group. Samples were administered orally once and observed for side effects or lethality for 7 days after administration. That is, on the day of administration, changes in general symptoms and the presence or absence of dead animals were observed at least 1 hour, 4 hours, 8 hours, 12 hours after the administration, and at least once in the morning and afternoon every day from the day after the administration.

As a result, all mice treated with all the samples obtained in Example 2 did not show any clinical symptoms, no mice died, and no toxicity was observed in weight changes, blood tests, blood biochemical tests, autopsy findings, and the like. No change was observed.

Therefore, the compound of the present invention did not show a toxicity change up to 1,000 mg / kg in mice, it was confirmed that oral administration minimum dose (LD ₅₀ ) is a safe substance of 1,000 mg / kg or more.

Examples of pharmaceutical preparations or health foods containing licorice extract, fractions, or compounds isolated therefrom or salts thereof will be described.

Manufacturing example  1: Preparation of Pharmaceutical Formulations

1-1. Powder  Produce

Licorice extract, fractions, or compounds isolated therefrom or 2 g

1 g lactose

After mixing the above components, the airtight cloth was filled to prepare a powder.

1-2. Manufacture of tablets

Licorice extract, fraction, or compound isolated from it or salt 100 mg

100 mg corn starch

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

1-3. Preparation of Capsules

Licorice extract, fraction, or compound isolated from it or salt 100 mg

100 mg corn starch

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

1-4. Preparation of Injection

Licorice extract, fractions, or compounds isolated therefrom or 10 μg / ml

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

Dissolve licorice extract, fraction, compound isolated from it, or salt thereof in an appropriate volume of sodium chloride BP for injection, adjust the pH of the resulting solution to pH 3.5 using dilute hydrochloric acid BP, and use sodium chloride BP for injection. The volume was adjusted to sufficiently mix. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

Manufacturing example  2: manufacture of health food

2-1. Preparation of Cooking Seasonings

Licorice extract, fractions, or 0.2 ~ 10% by weight of a compound or salt thereof prepared from the health promotion cooking seasoning was prepared.

2-2. Preparation of Tomato Ketchup and Sauce

Licorice extract, fraction, or 0.2 to 1.0% by weight of a compound or salt thereof separated from the tomato ketchup or sauce was prepared to prepare a health promotion tomato ketchup or sauce.

2-3. Manufacture of flour food products

Licorice extract, fraction, or 0.1 to 5.0% by weight of the compound or salt thereof separated from the flour was added to the flour, and the bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.

2-4. soup  And juicy ( gravies Manufacturing

Licorice extract, fractions, or 0.1 to 1.0% by weight of a compound or salt thereof separated from the liquor was added to soups and broth to prepare health products, noodles soup and broth.

2-5. ground Beef  ( ground beef Manufacturing

Licorice extract, fractions, or 10% by weight of a compound or salt thereof separated from the ground beef was added to ground beef to prepare a ground beef for health promotion.

2-6. dairy product ( dairy products Manufacturing

Licorice extract, fractions, or 0.1 to 1.0% by weight of a compound or salt thereof separated from the liquor was added to milk, and the milk was used to prepare various dairy products such as butter and ice cream.

2-7. Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried in a known manner, and then roasted to prepare a powder having a particle size of 60 mesh.

The licorice extract, fractions, or the compounds or salts thereof separated from the licorice extract were concentrated under reduced pressure in a vacuum concentrator, and dried by spraying and drying with a hot air dryer. The dried product was ground to a particle size of 60 mesh to obtain a dry powder.

The grains, seeds and licorice extracts, fractions, or dry powders of the compounds or salts thereof prepared above were prepared by blending in the following ratios.

Cereals (30% brown rice, 15% barley, 20% barley),

Seeds (7% by weight perilla, 8% by weight black beans, 7% by weight black sesame seeds),

Licorice extract, fraction, or dry powder (1% by weight) of a compound or salt thereof separated therefrom,

Ganoderma lucidum (0.5% by weight),

Foxglove (0.5 wt%)

2-8. Preparation of Carbonated Drinks

Syrup was prepared by mixing 5-10% of sugar, 0.05-0.3% citric acid, 0.005-0.02% caramel, 0.1-1% vitamin C, and 79-94% purified water. The syrup prepared above was sterilized at 85 to 98 ° C. for 20 to 180 seconds, mixed with cooling water at a ratio of 1: 4, and then 0.5 to 0.82% of carbon dioxide was injected to extract licorice, fractions, or compounds thereof. Or carbonated drinks containing salts thereof were prepared.

2-9. Health drink  Produce

Homogeneous components such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), water (75%) and licorice extracts, fractions, or compounds or salts thereof separated from After sterilization and instant sterilization, it was packaged in small packaging containers such as glass bottles and plastic bottles to prepare healthy drinks.

2-10. Vegetable juice  Produce

Licorice extract, fractions, or 0.5 g of the compound or salt thereof isolated therefrom were added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

2-11. Fruit juice  Produce

Licorice extract, fractions, or 0.1 g of the compound or salt thereof isolated therefrom was added to 1,000 ml of apple or grape juice to prepare fruit juice for health promotion.

Claims (16)

A composition for preventing or treating rotavirus infection comprising licorice extract or fractions thereof as an active ingredient.
The method of claim 1, wherein the extract is a composition for preventing or treating rotavirus infection, characterized in that extracted from water, C ₁ ~ C ₄ alcohol or a mixed solvent thereof.
According to claim 1, wherein the fraction is a hexane fraction, ethyl acetate fraction or water fraction composition for the prevention or treatment of rotavirus infection, characterized in that.
According to claim 1, wherein the rotavirus is a human (Human), pig (Pocine), Bovine (Bovine), or Goat (Goat) rotavirus composition for the prevention or treatment of rotavirus infection, characterized in that.
The composition for preventing or treating rotavirus infection according to claim 1, wherein the rotavirus causes diseases of enteritis, diarrhea, cold, sore throat, bronchitis, and pneumonia.
According to claim 1, wherein the composition is a composition for the prevention or treatment of rotavirus infection, characterized in that the pharmaceutical composition, food composition or quasi-drug composition.
The method of claim 6, wherein the pharmaceutical composition is a tablet, pill, powder, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories A composition for the prevention or treatment of rotavirus infection, characterized in that it has any one formulation selected from the group consisting of.
The method of claim 6, wherein the food composition is from the group consisting of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soups, drinks, tea, drinks, alcoholic beverages and vitamin complexes A composition for the prevention or treatment of rotavirus infection, characterized in that any one selected.
According to claim 6, wherein the quasi-drug composition is used for the production of any one selected from the group consisting of disinfecting detergent, shower foam, gagreen, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment and filter filler. A composition for preventing or treating rotavirus infection.
A composition for preventing or treating rotavirus infection comprising any one of the compounds represented by Formulas 1 to 5 as an active ingredient.

[Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

The composition for preventing or treating rotavirus infection of claim 10, wherein the rotavirus is human, pig, bovine, or goat rotavirus.
11. The composition for preventing or treating rotavirus infection according to claim 10, wherein the rotavirus causes diseases of enteritis, diarrhea, cold, sore throat, bronchitis, and pneumonia.
The composition for preventing or treating rotavirus infection according to claim 10, wherein the composition is a pharmaceutical composition, a food composition or a quasi-drug composition.
The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is a tablet, pill, powder, granule, capsule, suspension, liquid solution, emulsion, syrup, sterilized aqueous solution, non-aqueous solvent, suspension, emulsion, lyophilized agent and suppository. A composition for the prevention or treatment of rotavirus infection, characterized in that it has any one formulation selected from the group consisting of.
The method of claim 13, wherein the food composition is from the group consisting of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soups, drinks, tea, drinks, alcoholic beverages and vitamin complexes A composition for the prevention or treatment of rotavirus infection, characterized in that any one selected.
The method of claim 13, wherein the quasi-drug composition is used in the manufacture of any one selected from the group consisting of disinfecting detergent, shower foam, gagreen, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment and filter filler. A composition for preventing or treating rotavirus infection.